Cystic fibrosis transmembrane conductance regulator (CFTR) protein

ABSTRACT

A substantially homogeneous protein having cystic fibrosis transmembrane conductance regulator activity is provided. Also provided is a therapeutically effective composition for treating a subject having cystic fibrosis.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/123,864, filed Sep. 20, 1993, which is a continuation ofU.S. application Ser. No. 401,609, filed Aug. 31, 1989, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 07/399,945,filed Aug. 24, 1989, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 07/396,894, filed Aug. 22, 1989, nowabandoned.

This invention relates to purified and functionally reconstitutedpreparations of Cystic Fibrosis Transmembrane Conductance Regulator(CFTR) and to pharmaceutical compositions and methods of use employingthese preparations.

The discovery of the gene which is mutated in patients with cysticfibrosis (CF) and the principal disease-causing mutation (Rommens etal., 1989; Riordan et al., 1989; Kerem et al., 1989) has given rise tothe possibility of the development of molecular therapies. These can beconsidered in at least three broad categories: A.) The creation oridentification of drugs to appropriately modify CFTR function orbiosynthesis; B.) gene therapy by the delivery of the CFTR DNA sequencein an appropriate vector to affected epithelial cells; C.) proteinreplacement therapy in which the CFTR protein in an appropriate vehicleis delivered to the same cells.

The steps to be accomplished for the effective application of the thirdstrategy 1) the production of large quantities of functional CFTRprotein; 2) the solubilization and purification of the CFTR protein; 3)the reconstitution of the homogeneous purified protein into a lipidenvironment in which it can function; 4) demonstration that the purifiedand reconstituted CFTR molecule has the same functional properties as ithad in the epithelial cells to which it is native; 5) fusion ofproteoliposomes containing functional purified CFTR with the apicalsurfaces of CF epithelial cells expressing nonfunctional mutant CFTR orno CFTR at all in order to restore regulated chloride channel activity.

The original CFTR cDNAs which we had isolated and cloned (Riordan etal., 1989) and deposited with ATCC have been used for expression of CFTRin a number of different heterologous mammalian cell systems (Tabcharaniet al., 1991; Anderson et al., 1991a; Cheng et al, 1990; Dalemans etal., 1991). However, because of limitations on the amount of CFTR whichcan be synthesized in human and other mammalian cells (Cheng et al.,1990; Gregory et al., 1991), it was necessary to utilize an alternativesystem to generate adequate amounts for purification. We employed thebaculovirus expression vector system (BEVS; Lucknow and Summers, 1988)to produce large quantities of functional human CFTR in insect Sf9 cells(Kartner et al., 1991). More recently, others have produced CFTR proteinin the milk of transgenic mice (DiTullio et al., 1992) as anotherpotential means of producing sufficient protein for purification.However, in that work no evidence of functionality was demonstrated, norwere any attempts at purification made.

The present invention involves the fulfilment of steps 2.), 3.) and 4.)resulting in the production of highly purified CFTR protein as judged bystringent criteria of homogeneity. The purified protein is furtherdemonstrated to exhibit the same functional properties of a regulatedchloride ion channel as it does in its native location in vivo. Inaddition, as expected, structural features including N-terminal aminoacid sequence (6 residues), overall amino acid composition andisoelectric point are identical to those predicted from the translatedDNA sequence of the coding region of the cloned CFTR gene. The onlyfeature of the protein produced in the insect cell expression systemwhich differs from that produced in human epithelial cells in the typeof carbohydrate added when the protein is glycosylated during synthesis.However, we have already demonstrated that this difference is withoutinfluence on the function of the glycoprotein (Kartner et al., 1991).The glycosylation of the protein in any other of the alternateexpression systems which may be used such as milk of transgenic animals(DiTullio et al., 1992) will also differ from that in the human lungwhich will be the principal site of delivery for therapeutic purposes.

The invention also teaches that the proteoliposomes of the type known tobe capable of fusing with the membranes of cells, can be fused to planarlipid bilayers in which the generation of electrical currents carried bychloride ions through the CFTR channel can be measured.

In accordance with a further aspect of the invention, there is provideda means of replacement of defective CFTR in the epithelial cells from CFpatients by delivery to them of the purified and reconstitutedrecombinant CFTR protein.

In addition to its direct use in protein replacement therapy, thepurified CFTR of the invention provides for the development ofalternative therapeutic strategies, for example the development ofrationally designed drugs based on features of the molecule's structurewhich can be determined from the purified preparation.

BACKGROUND OF THE INVENTION

The cloning of the gene mutated in patients with cystic fibrosis (CF)has made possible interpretation of the deduced primary structure of thegene product, CFTR. In the context of what was known of an epithelialCl⁻ permeability defect in CF, this lead to the original suggestion thatthe gene coded for either a Cl⁻ channel itself or a regulator of aseparate Cl⁻ channel (Riordan et al, 1989). The introduction ofexpressible CFTR cDNAs into cells bearing CF-causing mutations in thegene (Rich et al, 1990; Druman et al, 1990), or into cells in which CFTRis not normally expressed (Anderson et al, 1991a; Kartner et al, 1991;Rich et al, 1991; Bear et al, 1991; Dalemans et al, 1991; Drumm et al,1991) resulted in the appearance of a Cl⁻ conductance regulated bycyclic AMP and similar to that seen in several normal epithelial celltypes (Gray et al, 1989; Tabcharani et al, 1990). A low conductanceohmic Cl⁻ channel activated by protein kinase A (PKA)--catalysedphosphorylation and inactivated by dephosphorylation was shown tounderlie this conductance pathway (Tabcharani et al, 1991; Berger et al,1991). Although these findings cannot distinguish between the CFTRprotein constituting the conductance pathway itself, or its being aphosphorylation-activated regulator, changes in ion selectivity onmutation of amino acids with charged side chains in the proposedtransmembrane sequences (viz. K95 in TM1 and K335 in TM6; Anderson etai, 1991b) tend to support the former possibility.

Consistent with its proposed role as an ion channel, CFTR is arelatively non-abundant protein in the epithelial tissues in which it isendogenously expressed. We know of no tissue which provides an adequatesource for purification. Similarly, it has not yet been possible toestablish mammalian cell lines in which a very high level ofheterologous expression of CFTR occurs (Cheng et al, 1990). This isbelieved to be at least partially due to a rather stringent control ofCFTR biosynthesis which limits the amount of wild type protein whichaccumulates in cells (Gregory et al, 1991). This quality control isapparently more strictly enforced in the case of some mutant forms ofCFTR, including the product of the most common mutation (F508), in whichlittle or no mature protein is detectable and only small amounts ofimmature precursor is present, apparently in the endoplasmic reticulum(Cheng et al, 1990).

Until the work of the present inventors, no one had succeeded inisolating CFTR and purifying it to substantial homogeneity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C. Enrichment of CFTR during major steps of purification.Aliquots containing approximately 1 μg of protein were subjected toSDS-PAGE (6% acrylamide in A and B and 4-15% acrylamide in C). Panels Aand C show results of silver staining and B is an immunoblot probed withmonoclonal antibody M3A7. In 1A and 1B, lane 1 is the initial crudeparticulate fraction; lane 2, the same fraction after alkali extractionof peripheral membrane proteins; lane 3, the highly enriched peak `F`from hydroxyapatite; lane 4, the CFTR-containing fraction of the finalSuperose 6 step. In panel C only the final gel filtration purifiedfraction was run.

FIGS. 2A-2B. Major purification of CFTR by hydroxyapatitechromatography. The upper panel shows the elution profile with phosphategradient indicated and the lower panel shows silver staining proteinbands after SDS-PAGE of fractions as indicated. Lane F containing mostof the CFTR clearly corresponds to lane 3 in panels 1A and 1B of FIG. 1.

FIG. 3. Gel filtration chromatography on Superose 6 of peak F from FIG.2. Fractions constituting peak 1 contain highly purified CFTR asindicated in lane 4 of FIG. I A and B, table II and N-terminal sequenceanalysis.

FIGS. 4A-4B. 2-D gel electrophoresis of purified CFTR. For isoelectricfocusing 2% ampholytes ranging from pI 3.5 to 10 and 7% acrylamide wasused. Electrophoresis was as with 1-D gels as was silver staining (4A)and immunoblotting (4B).

FIG. 5. Electronmicroscopy of negative stained proteoliposomescontaining CFTR. Vesicles on carbon formvar coated grids were stainedwith 2% uranyl acetate. Scale is 0.1 μm.

FIGS. 6A-6C. Low Conductance Cl⁻ Channel Associated with CFTR Expressionin Sf9 Cells. (6A) This panel shows PKA-activated single channel currenttracings at various pipette potentials in excised patches. The pipetteand the bath contained symmetrical salt solutions (NaCl=140 mM). Inaddition, PKA (200 nM) and ATP (1 mM) were added to the bath tostimulate the appearance of this channel. (6B) Currents tracings fromPKA-stimulated channels in excised membrane patch from CFTR-expressingSf9 cell. In this case, channels were studied in the presence of 300 mMNaCl in the bath and 50 mM NaCl in the pipette. (6C) Current-voltagerelationships are shown for PKA-stimulated channel in symmetrical NaClsolutions (bath and pipette=140 mM) (o) (n=8) and asymetrical NaClsolutions (bath=300 and pipette=50 mM NaCl) (•) (n=9). Means and SD havebeen shown. Arrows indicate the closed conductance state.

FIG. 7. Purified CFTR functions as a phosphorylation activated ionchannel in lipid bilayers (a) The upper trace shows four nystatin spikes(o) which fail to lead to single channel activity. Two nystatin spikesare long lasting and two short lasting, the differences possiblyreflecting stochastic variation in the number of nystatin units perliposome. Scale bars indicate 1 pA vertically and 5 sec horizontally.The lower trace shows a short lasting nystatin spike which is followedby the appearance of single chloride channel activity. PKA and Mg-ATPare present in both the cis and trans compartments. The scale barsindicate 1 pA vertically and 2 sec horizontally. Holding potential is-25 mV. (b) Addition of liposomes which do not contain purified CFTR tobilayer chamber with PKA (200 nM) and Mg₂ ATP (1 mM) fails to causeappearance of unitary current steps. (c) Addition of liposomescontaining CFTR with no added PKA and Mg₂ ATP fails to evoke singlechannel activity. (d) Single channel activity is apparent only in thoseexperiments in which CFTR-containing liposomes are added to the bilayerchamber with PKA and Mg₂ ATP. The applied potential was -25 mV in thisexperiment.

FIGS. 8A-8D. Comparison of current-voltage relationship ofPKA-stimulated purified CFTR and Cl⁻ channel in T84 cells andCFTR-expressing CHO cells. (8A) Current traces are shown for purifiedCFTR protein in lipid bilayer at various potentials. The cis compartmentof bilayer chamber contained 300 mM KCI, PKA (200 nM) and Mg-ATP (1 mM).The trans compartment contained 50 mM KCI+PKA and Mg-ATP. (8B) I-Vrelationships are shown for conductance of PKA-activated purifiedprotein with 300 mM KCI in the cis compartment and 50 mM KCI(.increment.) (n=6) or 10 mM KCI (o) (n=4) in the trans compartment.(8C) The upper panel shows two 11 pS channels opening sequentially instepwise manner. The lower panel shows a larger conductance observed inthe same recording which corresponds to twice the conductance of themore prevalent smaller conductance and may represent cooperative gatingof two 11 pS channels. Holding potential was -45 mV. (D) I-Vrelationships for PKA-activated purified CFTR (∘) (n=4), PKA-activatedchloride channel in T84 (.increment.) (n=4) and PKA-activatedCFTR-expressing CHO (•) (n=4) membranes studied in planar lipid bilayers(cis:trans=300:10 mM KCl).

DESCRIPTION OF THE INVENTION

The inventors showed previously (Kartner et al, 1991) that large amountsof functional CFTR can be generated in Sf9 insect cells using thebaculovirus expression vector system (BEVS; Luckow and Summers, 1988),thereby providing the starting point for purification. Suspensioncultures of these cells have been used to obtain relatively largequantities of a crude CFTR-containing particulate fraction as startingmaterial for purification. Cold alkaline extraction (Steck and Yu, 1973)of the particulate fraction resulted in removal of approximately 2/3 ofthe total protein while retaining CFTR (Table I; FIG. 1A and B). At thisstage a CFTR band was clearly visible by protein staining followingSDS-PAGE (FIG. 1A). Our strategy for solubilization and furtherfractionation employed the strong dissociating conditions of an ionicdetergent for two principal reasons. First, systematic testing of theeffectiveness of a range of non-ionic and ionic detergents to solubilizeCFTR in membranes of T84 epithelial cells, CHO cells or Sf9 cellsexpressing the protein showed that only the stronger ionic ones wereeffective. Second, because our major aim was to determine whether CFTRcould function as a regulated Cl⁻ channel we wished to minimize thepossibility of copurification of any proteins which might associate withCFTR and contribute to the function of the final purified material.

Conventional column chromatography techniques compatible with thepresence of sodium dodecyl sulfate (SDS) (at 0.15 to 0.25%) were thentested for their ability to separate CFTR from other proteins. Among themethods attempted, adsorption to hydroxyapatite proved to be by far themost effective (FIG. 2). CFTR interacted with the matrix more stronglythan nearly all other proteins under the conditions employed and waseluted only after the phosphate gradient had reached its maximumconcentration of 600 mM. A purification of at least one hundred fold wasachieved in this step (Table 1). A major contaminating protein ofapproximately 30 kd, and very high molecular weight material running atthe origin of a 6% acrylamide gel, was also present in this fraction.Minor amounts of faintly silver staining material at molecular weightsboth lower and higher than the principal CFTR band were also detectable;the immunoblot of this lane indicates that at least some of these aredegraded and aggregated forms of CFTR, respectively. CFTR could beseparated from the remaining contaminants by gel filtration on Superose6 (FIG. 3). The protein eluted in a well-resolved included peakcorresponding to 28% of the Superose gel volume. From one liter of cells(5×10⁹) approximately 0.5 mg of CFTR protein was obtained in this peak.

Characterization of purified CFTR

The effectiveness of the major purification steps is summarized in FIG.IA and B. The presence in the final product assessed on 6% SDS-PAGE gelsof a single silver staining band reactive with monoclonal antibodies toCFTR indicates that it is not contaminated with other proteins largerthan the cutoff molecular weight of the gel. To determine if stillsmaller proteins might be present a 4-15% gradient gel was heavilyloaded and stained with silver (FIG. IC). No other bands weredetectible.

As a more stringent assessment of homogeneity, two dimensional gelelectrophoresis (Dottin et al, 1979) was performed and analysed bysilver staining and immunoblotting (FIG. 4). As in the 1-D gels nocontaminating proteins were detected. The major isoelectric form of CFTRmigrated at a position corresponding to a pI of approximately 9. Thisagrees well with the value of 8.98 calculated from the CFTR amino acidcomposition. Since only core, mannose-containing N-linkedoligosaccharides are added to proteins in Sf9 cells (Vialand et al,1990) carbohydrate would not be expected to influence the pl. The smallimmunoreactive spots may represent alternate isoelectric forms of CFTR.

The final purified protein was subjected to both N-terminal sequencedetermination and quantitative amino acid compositional analysis. Thesequence of the N-terminal 6 residues agreed with that predicted fromthe DNA sequence for CFTR and there was excellent agreement between theamounts of each residue in the overall composition. The amino acidcomposition compared with that deduced from the sequence is shown intable II.

CFTR Reconstitution into phospholipid vesicles

In order to be able to determine the functional capacity of purifiedCFTR it was necessary to transfer the detergent solubilized protein backinto a lipid environment. This was done by a dialysis protocol analogousto that employed for renaturation of bacteriorhodopsin (London andKhorana, 1982; Braiman et al, 1987) and some other transport proteins.Essentially, pure CFTR in 0.25% lithium dodecyl sulfate (LIDS) was mixedwith a sonicated phospholipid mixture (PE:PS:PC at 5:2:1) containing 1%cholate and dialysed extensively to form proteoliposomes. Followingdispersion by sonication, and concentration, these proteoliposomes werefused with liposomes of the same phospholipid composition but alsocontaining ergosterol and nystatin to promote and enable detection ofsubsequent fusion to planar lipid bilayers (see below). Thismodification to enable the nystatin-induced liposome fusion was takenfrom Woodbury and Miller (1990). Following elution in the void volume ofa Superose 12 gel filtration column, all of the CFTR employed in thereconstitution could be accounted for in immunoblots of theproteoliposomes.

Negative staining indicated a uniform population of CFTR containingproteoliposomes, about half of which have a diameter of 40-60 nm withthe other half at approximately 15-20 nm (FIG. 5). Some fused largervesicles were also present. The 5 nm vesicles would have a surface areaof 7.9×10⁵ Å². Using a value of 50 Å² for the average area occupied by aphospholipid molecule (Levitzki, 1985) there should be 1.6×10⁴phospholipid molecules per vesicle. Since we used 2 mg of phospholipidor 1.5×10¹⁸ molecules approximately 10¹⁴ vesicles will have formed.These had incorporated 6.38 μg (based on quantitative amino acidanalysis) corresponding to 3.8×10⁻¹¹ moles or 2.3×10¹³ molecules ofCFTR. Therefore, not more than one (approximately 0.23) CFTR hadincorporated per 50 nm vesicle. The number of these vesicles which weresubsequently fused to a black lipid film could then be monitored by thenystatin mediated conductance spikes (see below).

Effect of CFTR-containing proteoliposomes on a planar bilayer

A cyclic AMP-activated, low-conductance chloride channel has beendescribed in CFTR expressing Sf9 cells (Kartner et al., 1991). In orderto compare the conductive properties of purified CFTR with that of theCFTR-associated channel in native membranes, it was first necessary tocharacterize this channel in Sf9 cell membranes under the sameconditions which would later be dictated by the requirements of theplanar bilayer. These conditions include an ionic gradient which isfavorable for liposome fusion, i.e. the presence of an osmotic gradient(300 mm KCl versus 50 mm KCl). Therefore, the low conductance Cl⁻channel conductance in excised membrane patches from CFTR-expressing Sf9cells was compared in symmetric and asymmetric ion gradients in order toassess the influence of these gradients on single ion conductance (FIG.6).

No single channel activity corresponding to that of the low-conductanceCl⁻ channel was detected in excised patches from CFTR-expressing Sf9cells unless PKA (200 nM) and Mg-ATP (1 mM) were added to the bath. Withthe addition of PKA, a nonrectifying, 10.1 pS channel was detected with140 mM NaCl in both the patch pipette and the bath (n=39). Thisobservation supports the previous description of PKA regulation of thesmall, nonrectifying Cl⁻ channel in excised patches of CHO cellsexpressing CFTR (Tabcharani et al., 1991). In the presence of an iongradient comparable to that required for liposome fusion in planarbilayer studies, ie. 300 mM NaCl in the bath and 50 mM NaCl in the patchpipette (n=19), the current voltage (I-V) relationship of thePKA-regulated channel showed a shift in reversal potential toapproximately 50 mV, a change consistent with high chloride selectivity,and an increase in unitary conductance to 14.1 pS. This relatively smallchange in conductance from 11 to 14 pS with a two fold increase inchloride concentration suggests that the effect of chlorideconcentration on unitary conductance is nonlinear. Tabcharani andHanrahan have found that in excised patches from CHO cells expressingCFTR, the low conductance Cl⁻ channel saturates as a function of Cl⁻activity with a Michaelis-Menten Km in the range of 35 to 40 mM.

In initial experiments with planar lipid bilayers, the CFTR-containingproteoliposomes were simply added to the cis chamber with mixing.Although indications of the appearance of Cl⁻ channel activity weredetected early on, the fusion frequency was apparently low because thecurrent changes eventually found to be characteristic of this channel inthe bilayer were only observed infrequently (in 3 of 15 experiments)even in the presence of ATP and PKA. This made it difficult to be sureof the significance of the lack of activity under non-activatingconditions. Therefore, we sought a means of both promoting and detectingfusion events. The nystatin fusion technique described just a yearearlier by Woodbury and Miller (1990) seemed as if it should suit thispurpose and was attempted. The rationale is that the ergosterol-nystatincomplex which is incorporated into phospholipid vesicles during theirformation provides non selective ionophore activity, thus generating anionic and osmotic gradient which promotes fusion of the vesicles withthe bilayer. When this occurs, a transient current spike is observedproviding an index of the fusion events. Because nystatin rendersessentially all vesicles fusogenic, the channel activity observed isrepresentative of the whole population of vesicles. Since we hadquantified the amount of CFTR in our vesicles, this technique provided ameans of evaluating how much of it entered the bilayer. FIG. 7A shows acurrent tracing containing these spikes and the coincidence of a lowconductance Cl⁻ current with one of them. On average this occurs once in10 and 20 spikes. Since we had calculated that approximately one in fourlipid vesicles contained a CFTR molecule after reconstitution, itappears that 20-40% of the purified protein molecules are capable ofgenerating a channel in the bilayer.

FIGS. 7 B, C and D show records representative of many experiments toassess the relationships of the properties of the channel assayed inthis way to those exhibited in the patch-clamp experiments with thecells from which CFTR had been purified. Fusion of liposomes withoutadded CFTR (FIG. 7B) failed to produce the appearance of stepwisechanges in current levels in the presence of PKA and Mg-ATP (added toboth cis and trans compartment in all cases; n=6). Furthermore, fusionof CFTR-containing liposomes without added PKA and Mg-ATP (FIG. 7C)failed to evoke the appearance of single channel currents in 35experiments in which fusion was achieved. Similarly, the addition of ATPalone, prior to PKA addition did not cause the appearance of singlechannel steps. Switch-like transitions in current level were onlydetected following fusion of CFTR containing liposomes in the presenceof both PKA and ATP (FIG. 7D). Single channel events were observed in 35of 45 experiments in which nystatin-induced fusion spikes were observed.Hence, in these experiments approximately 550 independent fusion eventswere detected and as a consequence 35 low conductance single channelevents were detected. No single channel currents were detected in 10 ofthese 45 experiments due to problems of high noise and bilayer breakage.The mean open probability of the PKA stimulated channel was 0.38±0.13for five experiments, a value close to that reported by Tabcharani et al(1991) for phosphorylation activated Cl⁻ channel in excised patches fromCFTR-expressing CHO cells of 0.41.

The current-voltage relationship of the reconstituted CFTR protein wasfound to be comparable to that observed for CFTR-expressing Sf9membranes studied by the patch-clamp technique in the presence ofsimilar ionic gradients. The slope conductance associated with thepurified protein was 11.1 pS in the presence of 300 mM KCI in the ciscompartment and 50 mM KCI in the trans compartment (FIGS. 8A and B). Weobserved no marked effect of voltage on channel open probability, acharacteristic shared with the PKA-activated Cl⁻ channel studied inCFTR-expressing CHO cells (Tabcharani et al, 1991). The anion versuscation selectivity was estimated from the reversal potentials of the I-Vcurves. With 300 mM KCI on the cis side and 50 mM KCI on the trans side,one expects a reversal potential of 45 mV for an ideally anion selectivechannel. The extrapolated value obtained from 6 experiments was 39 mV.Furthermore, in the presence of the gradient (cis:trans=300:10) it isexpected that current will reverse at 86 mV for an ideally anionselective conductance path. The extrapolated value from four experimentswas 79 mV. We estimated using the Goldman-Hodgkin-Katz equation,therefore, that Cl⁻ /K⁺ selectivity is at least 10:1. High chlorideselectivity is another feature this channel shares with theCFTR-associated channel studied in CHO cells (Tabcharani et al, 1991).

Occasionally, in 4 of 35 experiments, another conductance level wasdetected in addition to the 11 pS conductance. This relatively rareconductance level corresponded to twice that of the predominant one andwe believe that the larger conductance reflects cooperative gating oftwo 11 pS channels (FIG. 8C). Cooperative gating between differentconductance states has been described for several purified channels,including the acetylcholine receptor (Schindler et al., 1984), bacterialporin (Engel et al, 1985) and the ryanodine receptor (Smith et al.,1988).

The identity of the conductance caused by purified CFTR incorporationinto lipid bilayers with that present in cells which endogenouslyexpress CFTR, ie. T84 cells, and that conductance conferred by CFTRexpression in CHO cell membranes, was confirmed in the experiments shownin FIG. 8D. The I-V relationships of the channels activated by PKA andATP addition to purified CFTR, T84 and CFTR-containing CHO membranesfollowing fusion with the lipid bilayer are virtually superimposable,showing similar unitary conductances (11.1 pS, 10.0 pS and 9.23 pSrespectively) and reversal potentials (80 mV, 84 mV and 89 mV,respectively) in the presence of a 300/10 KCI gradient. Significantly,this PKA-activated chloride conductance was not observed followingfusion of plasma membranes prepared from untransfected CHO cells withlipid bilayers. Only a relatively large conductance, approximately 40pS, chloride channel was observed consistently when untransfected CHOcell membranes were used, but this channel was active both in thepresence and in the absence of PKA. An identicalphosphorylation--independent channel was also observed inCFTR-containing CHO cell membranes. This 40 pS channel is similar withrespect to its conductance and rapid kinetics to that described byReinhardt et al (1987) in bilayer studies of rat colonic apicalmembranes.

The inventors have demonstrated that a regulated Cl-channel withproperties similar to that observed in intact cells can be detected inplanar lipid bilayers into which highly purified CFTR is incorporated.The CFTR protein has been successfully removed from the membrane,manipulated extensively and returned to a functional state.

To address the question of the postulated Cl⁻ channel activity of CFTR,quantitative considerations are required. A channel activation wasobserved in black lipid films approximately once for every ten to twentynystatin spikes reflecting the fusion of 10-20 vesicles. About one infour vesicles would contain a CFTR molecule (assuming a monomer althoughwe have no evidence of this). This suggested that 20-40% of thereconstituted CFTR molecules may be capable of activation. This is anexcellent degree of functional reconstitution given the type ofsolubilization, purification and reconstitution scheme used. However,these data also indicate that if channel activation required a proteinother than CFTR it would have to be present in at least one copy per 5CFTR molecules. The one and two dimensional gel electrophoresis data(FIGS. 1, 2 and 4) preclude the presence of 20% contamination.Furthermore, the fact that the purified protein yielded N-terminal aminosequence and overall amino acid composition indicative of the presenceof only CFTR would argue that any contaminants which may be present mustbe in very low amounts, indicating that the protein of the invention issubstantially homogeneous. Hence, we conclude that it is very likelythat regulated channel activity is a property of the CFTR moleculeitself. This invention presents the first functional characterisation ofa purified epithelial channel.

Having the protein in isolation from others will assist, for example, indetermining specific sites of interaction of modifying drugs such assulfonyl ureas which in a preliminary report have recently been claimedto influence CFTR (Sheppard and Welsh, 1992).

In Cystic Fibrosis patients, the epithelial cells of many tissues,especially those of the lining of the airways, either lack CFTR in thecell membrane or have nonfunctional variants of the protein.

The isolation and purification of the CFTR protein, as in the presentinvention, makes possible therapy for Cystic Fibrosis patients, byrestoring functional CFTR protein to the epithelial cells of the airwaysof the lung.

Proteins can be incorporated into cell membranes when they are suppliedto the cell surface in association with a suitable carrier which assiststhe protein to be incorporated into the cell membrane, where it restoresfunction. Suitable carriers will be known to those skilled in the artand include lipid vehicles such as the proteoliposomes of the presentinvention, which fuse with the cell surface allowing their contents tobe incorporated into the cell membrane.

The protein plus carrier is administered to the epithelial cells to betreated by conventional means, for example by aerosol delivery to theairways of the lung.

Additional agents may be incorporated into the proteoliposomes toimprove targeting towards a particular tissue, for example antibodies toparticular cell surface molecules may be incorporated.

EXAMPLE 1 Cell Culture

Sf9 insect cells were infected with a recombinant Baculovirus containingthe complete CFTR coding sequence as described previously (Kartner etal., 1991). For patch-clamp experiments cells were grown attached toplastic tissue culture dishes in Grace's media. For the purposes ofprotein purification, cells were gown in suspension culture. The humancolonic carcinoma-derived epithelial cell line T84 (Dharmsathaphorn etal., 1984) was grown on a plastic substrate in 1:1 of Dulbecco'smodified Eagle's medium and F-12 culture medium, and CHO cellsexpressing CFTR (Tabcharani et al, 1991) were grown in alpha modifiedminimal essential medium. All of these culture media were supplementedwith 5 to 10% fetal bovine serum. Membrane preparations from T84 cellswere obtained as described previously (Kartner et al, 1991) and highlypurified plasma membrane vesicles were isolated from CHO cells accordingto Riordan and Ling (1979).

CFTR Purification

Sf9 cells from one liter of suspension culture were collected 3 daysafter infection yielding a 5 ml pellet which was resuspended andhypotonically swollen in 50 ml of 18 mM KCl, 5 mM sodium citrate, pH 6.8(containing phenylmethyl sulfonyl fluoride at 100 μg/ml; aprotinin andleupeptin at 50 μg/ml). Cells were disrupted with a Potter-Elvejhamhomogenizer, particulates pelleted and treated with DNAase I (1 μg permg total protein).

Mild alkaline extraction was performed for 2 minutes on ice with 20volumes of 10 mM NaOH containing 0.1 mM EDTA.

The pelleted extracted material was dissolved in 10 mM phosphate buffer,pH 6.4 containing 2% mercaptoethanol and 2% SDS and was applied to acolumn (2.6×20 cm) of hydroxyapatite (Biogel HT, Biorad Laboratories)which had been preequilibrated with 10 mM phosphate buffer, pH 6.4containing 0.15% SDS and 5 mM dithiothreitol (DTT). After washing with50 ml of equilibration buffer, a 100 ml linear gradient (100 mM to 600mM) of sodium phosphate containing 0.15% SDS and 5 mM DTT was applied ata flow rate of 0.2 ml per minute. Elution was continued with the highphosphate buffer for an additional 100 ml at a flow rate of 0.1 ml perminute. Absorbance was monitored continuously at 280 nm and aliquots ofeach fraction were monitored for CFTR by dot blots on nitrocelluloseprobed with the monoclonal antibody M3A7. Positive fractions werefurther assayed by SDS-PAGE and immunoblotting.

CFTR-containing fractions from the hydroxyapatite column weretransferred to Centricon centrifugal microconcentrator tubes (Amicon)with a 30 kd cutoff, washed with 10 mM Tris-HCI, pH 8.0 containing 100mM NaCl and 0.25% LiDS (lithium dodecyl sulfate) also in these devicesand again concentrated to 400 μl. This volume was applied to a Superose6 preparative FPLC column (Pharmacia) pre-equilibrated with this samebuffer. Fractions eluted from the Superose 6 column were monitored aswith the hydroxyapatite column.

CFTR Protein Detection end Characterization

One dimensional SDS-PAGE was according to Laemmli (1970) using 6%acrylamide gels as described previously (Kartner et al, 1991) or 4 to15% gradient gels. Two dimensional gel electrophoresis was performedaccording to Dottrin et al (1979). Total proteins in either type of gelwere stained with silver (Merril et al, 1981). Immunoblotting was asdescribed previously employing a monoclonal antibody (M3A7) generatedagainst a fusion protein containing residues 1197-1480 of CFTR.

CFTR Reconstitution into Phospholipid Vesicles

100 μl of 15 mM HEPES, 0.5 mM EGTA, pH 7.4 containing 1 mg of asonicated phospholipid mixture (PE:PS:PC/5:2:1) and 2% sodium cholatewas added to 100 μl of 10 mM Tris-HCl, pH 8.0 containing 100 mM NaCl,0.25% LiDS and 5 μg of purified CFTR. After a one hour incubation on icethe mixture was dialysed at 4° C. against 2 liters of the HEPES-EDTAbuffer for 5 days with daily buffer changes. The sample was furtherdialysed for 3 days against daily changes of 2 liters of the same buffercontaining 150 mM NaCl. The proteoliposomes thus obtained were sonicatedfor 15 seconds in a bath sonicator from Lab Supplies Co. Inc.,Hicksville, N.Y. 11801 (Model Gl128PlG) and concentrated to 100 μl in aMinicon B15 concentrator (Amicon). To introduce nystatin according tothe procedure of Woodbury and Miller (1990), this 100 μl sample wasmixed with 100 μl of protein-free liposomes which had been prepared bysonicating a mixture of PE:PS:PC:Ergosterol at a ratio of 5:2:1:2 (10mg/ml) in the presence of 120 μg/ml nystatin in the HEPES-EGTA buffercontaining 300 mM NaCl. The mixture was frozen and thawed and sonicatedfor 15-20 sec. This cycle was repeated and the final proteoliposomeseither used immediately for fusion with planar bilayers or frozen at-85°. In the latter case aliquots were thawed and sonicated brieflybefore use. The presence of intact CFTR was verified by exclusion from aSuperose 12 column (Pharmacia).

For estimation of their diameters, the proteoliposomes were pipettedonto carbon formvar-coated grids, then negatively stained with 2%aqueous uranyl acetate and viewed and photogaphed by transmissionelectron microscopy.

Incorporation of CFTR into Planar Bilayers

A 10 mg/ml solution of phospholipid (PE:PS at a ratio of 7:3) (AvantiPolar Lipids) in n-decane was painted over a 200 μm aperature in abilayer chamber to raise phospholipid bilayers. Bilayer formation wasmonitored electrically by observing the increase in membranecapacitance. In all experiments, bilayer capacitance was greater than200 pF.

The solution in the cis compartment (where proteoliposomes were added)typically contained 300 mM KCl, 10 mM MOPS, 1 mM MgCl₂ and 2 mM CaCl₂,pH=6.9. The trans solution contained 50 mM KCl, 10 mM MOPS, 1 mM MgCl₂and 2 mM CaCl₂, pH 6.9. Single channel currents were detected with apatch-clamp amplifier, modified for bilayer studies (Warner Instruments)and recorded following digitization (PCM2, Medical Systems) using a VCR.For playback of records, a 6-pole Bessel filter was used (100 Hz).Single channel current amplitudes were determined by the generation ofamplitude histograms using pCLAMP software. Open-state probability wasdetermined using the same software program and openings were definedusing the 50% threshold criterion.

Addition of 4 μl of proteoliposomes preparation to the cis compartmentof the bilayer chamber, followed by stirring (approximately 10 min)typically resulted in the appearance of 10-20 abrupt conductance stepsor spikes, indicative of fusion of roughly 10-20 liposomes with thelipid bilayer. The conductance steps are due to current flow throughergosterol-dependent nystatin channels and the transient nature of thesesteps reflects the dissociation of the ergosterol-nystatin complex asthe liposome composition diffuses into the ergosterol-free bilayer.Cessation of stirring prevented further liposome fusion with no furtherappearance of spikes. The incorporation of a channel with liposomefusion was detected as a stepwise change in current level. Membranepotentials were referenced to the trans compartment and Cl⁻ current fromcis to trans designated as negative.

Patch-Clamp Studies of CFTR-expressing cells

Single channel currents were recorded using conventional proceduresaccording to Hamill et al (Hamill et al, 1981) with a List EPC-7patch-clamp amplifier (Medical Systems, Great Neck, N.Y.) Pipettes werefabricated from borosillicate glass type 7052 (Gamer Glass Co.) using atwo-stage Narishige pipette puller. The bath electrode was a Ag-AgClwire connected to the bathing solution via an agar bridge. Currentoutput was monitored on a Tektronix oscilloscope and stored on videotapeafter A/D conversion by a video adaptor (PCM 2, Medical Systems). Singlechannel current records stored on video tape were transferred to thehard disk of an EBM-AT compatible computer using the FETCHEX program ofpCLAMP (Version V) software (Axon Inst.) Records were sampled at 0.5-2.0kHz. During playback, single channel records were filtered using a6-pole Bessel filter set at 100 or 200 Hz low pass frequency. Singlechannel current amplitudes were obtained by examination of amplitudehistograms generated using the pCLAMP FETCHAN analysis program. The meanof the peak amplitude was taken as a measure of the unitary currentamplitude.

Solutions: In excised patch studies the standard bath and pipettesolutions contained 140 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucoseand 10 mM MES, pH 6.3. In some studies, the pipette solution contained50 mM NaCl plus sucrose (added to adjust osmolarity to 280 mOsm) and thebath solution contained 300 mM NaCl. Experiments were performed at22°-25° C.

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                  TABLE I                                                         ______________________________________                                        Protein Recovery during CFTR Purification                                                   Total protein                                                   Step          (mg)        Enrichment factor*                                  ______________________________________                                        Particulate pellet                                                                          300         1                                                   After alkali extraction                                                                     105         2.9                                                 After hydroxyapatite                                                                        1.0         300                                                 After Superose 6                                                                            0.5         600                                                 ______________________________________                                         Starting material was a one liter culture containing approximately 5          × 10.sup.9 cells.                                                       *assuming no loss of CFTR.                                               

                  TABLE II                                                        ______________________________________                                        Amino Acid Composition                                                                                       Ratio                                                                         Determined/                                    Residues Predicted*  Determined                                                                              Predicted                                      ______________________________________                                        Asx      113         100       0.88                                           Glx      160         154       0.96                                           Ser      122         127       1.04                                           Gly      84          84        1.00                                           His      24          24.4      1.01                                           Arg      78          76.4      .98                                            Thr      83          86.6      1.04                                           Ala      83          83        1.00                                           Pro      45          42        0.93                                           Tyr      40          nd                                                       Val      89          89.3      1.00                                           Met      38          36        0.95                                           Cys      18          20.8      1.15                                           Ile      120         113       0.94                                           Leu      18          181.2     0.98                                           Phe      84          82.8      0.99                                           Lys      92          96.8      1.05                                           ______________________________________                                         *from sequence                                                                nd  not determined                                                            1.5 μg of protein was hydrolysed with 6N HCl, PITC derivatized and         separated by HPLC on a PICOTAG column (Waters Associates).               

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A substantiallyhomogeneous protein having cystic fibrosis transmembrane conductanceregulator activity and characterised by migration as a single band onboth one- and two-dimensional gel electrophoresis.
 2. A protein inaccordance with claim 1 and having the determined amino acid compositionset out in Table II.
 3. A protein in accordance with claim 1 andspecifically recognized by monoclonal antibodies to cystic fibrosistransmembrane conductance regulator (CFTR).
 4. A protein in accordancewith claim 1 and having the N-terminal amino acid sequence: Met Gln ArgSer Pro Leu.
 5. A protein in accordance with claim i and having anempirically determined isoelectric point of approximately
 9. 6. Aprotein in accordance with claim 1 and having the ability to generate anohmic chloride channel having a unitary conductance of approximately 10to 14 pS when activated by protein kinase A.
 7. A protein in accordancewith claim 1 having a secondary, tertiary and quaternary structure whichcan be determined by physical biochemistry methods.
 8. A therapeuticallyeffective composition for treating a subject having cystic fibrosis byaerosol delivery of a therapeutic agent to the airways of the subject,said composition comprising a therapeutic agent comprising a protein inaccordance with any of claims 2 to 7 and a carrier suitable for aerosoldelivery of said protein to cells of the airways of said subject havingdeficient CFTR function.
 9. A therapeutically effective composition forthe treatment of cystic fibrosis, in accordance with claim 8, whereinthe composition may be delivered to a subject's airways.
 10. A methodfor treating a subject with cystic fibrosis comprising delivering atherapeutically effective composition in accordance with claim 8 to theairway passages of a subject by aerosol delivery.
 11. A therapeuticallyeffective composition for treating a subject having cystic fibrosis byaerosol delivery of a therapeutic agent to the airways of the subject,said composition comprising a therapeutic agent comprising a protein inaccordance with claim 1 and a carrier suitable for aerosol delivery ofsaid protein to cells of the airways of said subject having deficientCFTR function.
 12. A therapeutically effective composition in accordancewith claim 11 wherein said carrier is a lipid vehicle.
 13. Atherapeutically effective composition in accordance with claim 12wherein said carrier is a proteoliposome.
 14. A method for purifying arecombinant hydrophobic membrane protein comprising the steps of:(a)providing a sample of cells containing a membrane--associated protein tobe purified; (b) disrupting the cells and pelleting a particulatefraction thereof; (c) contacting the particulate fraction with a dilutealkali solution for an effective period of time at an effectivetemperature to extract unwanted constituents followed by repelleting theparticulate fraction; (d) dissolving the particulate fraction in abuffered denaturing detergent solution containing a thiol reducingagent; (e) subjecting the solution obtained by step (d) tohydroxyapatite chromatography to provide a partially purified protein;(f) subjecting the partially purified protein obtained by step (e) tomolecular sieve chromatography to provide a substantially homogeneousprotein; and (g) renaturing the protein obtained by step (f) bycompetitively removing sodium dodecyl sulphate from association with theprotein by exposure to excess sodium cholate and removing sodium cholateand sodium dodecyl sulphate by dialysis in the presence of phospholipidwhereby the protein is incorporated into phospholipid vesicles toprovide a functional protein.
 15. A method in accordance with claim 14wherein the membrane-associated protein is cystic fibrosis transmembraneconductance regulator and wherein step (d) comprises dissolving theparticulate fraction in 2% sodium dodecyl sulphate (SDS) and 2%mercaptoethanol in 10 mM phosphate buffer of pH 6.4, step (e) comprisesapplying the solution from step (d) to a hydroxyapatite columnpre-equilibrated with 10 mM phosphate buffer of pH 6.4 containing 0.15%SDS and 5 mM dithiothreitol (DTT), washing the column with the samebuffer and eluting the protein with a phosphate buffer gradientcontaining 0.15% SDS and 5 mM DTT; step (f) comprises chromatography ona Superose 6 preparative FPLC column in a suitable buffer containing0.25% lithium dodecyl sulphate (LIDS); and step (g) comprises combininga first solution containing the purified protein from step (f) in abuffer with a second solution containing a sonicated phospholipidmixture and 2% sodium cholate in a buffer, incubating the combinedsolution on ice for an effective period of time and dialysing thecombined solution against a buffer for an effective period of time togive proteophospholipid vesicles.